The present invention relates to 16-HETE analogs which are agonists and antagonists of 16-HETE. The invention also relates to methods and products for inhibiting neutrophil adhesion and neutrophil aggregation using the 16-HETE agonists.
The inflammatory response is an important element of a host""s natural defense mechanism against pathogens. It also is involved in wound healing. Despite the beneficial role that the inflammatory response plays in host survival, excessive inflammation may have clinically adverse results in some medical conditions.
Leukocytes are a major cellular component of inflammatory and immune responses. This class of cells includes neutrophils, lymphocytes, monocytes, eosinophils, and basophils. Neutrophils, which play a key role in the inflammatory response, are generally present within the body in a resting unstimulated state. Once stimulated, the neutrophils migrate to the site of injury and release toxic factors.
The migratory capability of a neutrophil is dependent on the ability of the neutrophil to alter its adhesive properties. In a resting unstimulated state a neutrophil is not adhesive and cannot migrate. Once the neutrophil has been stimulated, however, it becomes more adhesive and is capable of migrating. The increase in neutrophil adhesiveness causes the stimulated neutrophil to aggregate and to adhere to endothelium. Stimulation of the neutrophil also causes the neutrophil to undergo diapedesis, which involves the migration of the neutrophil between post-capillary endothelial cells into the tissues.
In the tissues, an activated neutrophil releases enzymes such as collagenase and elastase, among others. Neutrophil stimulation may also initiate a burst of oxygen consumption, with concomminant activation of the hexose-monophosphate shunt and activation of nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase. Activation of these systems results in the formation and release of factors such as hydrogen peroxide and hydroxyl radicals, which are toxic to microorganisms and tumor cells, and thereby facilitating the destruction of the injury causing agent.
Several studies have focused on analyzing the control and regulation of the adhesive properties of neutrophils. Much of this research has centered on adhesion receptors and also on metabolites of arachidonic acid such as C20 carbon fatty acid found in every cell membrane. Arachidonic acid metabolism occurs by different mechanisms in stimulated versus unstimulated neutrophils and results in the production of a different spectrum of metabolites in stimulated versus unstimulated neutrophils.
In stimulated neutrophils, the cytochrome P450 mixed function oxidase system appears to be more active. Moreover, during neutrophil stimulation, 5-lipoxygenase is translocated to the membrane compartment fraction, where it produces 5-hydroperoxyeicosatetraenoic acid (5-HPETE). 5-HPETE is then either metabolized to 5-hydroxyeicosatetraenoic acid (5-HETE) by peroxidase or dehydrated to form leukotriene A4. Leukotriene A4 is converted into leukotriene B4 which is a potent chemotactic agent and promoter of neutrophil adhesion.
In unstimulated neutrophils, the metabolism of arachidonic acid is markedly different than that is stimulated neutrophils. The metabolism of arachidonic acid in unstimulated neutrophils is sensitive to cytochrome P450 inhibitors but not to cyclooxygenase or lipoxygenase inhibitors. Hatzelmann and Ullrich characterized the metabolites produced in unstimulated neutrophils, reporting the finding that arachidonic acid is metabolized to 20-HETE and 15-HETE. Hatzelmann, Eur. J. Biochem. 173, 445-452 (1988). Another study, Kraemer et al., found that the arachidonic acid metabolic products formed in unstimulated neutrophils exhibited a potent anti-aggregatory activity toward human neutrophils, suggesting that the identified arachidonic acid metabolites may play some role in the regulation of neutrophil adhesion and aggregation properties. Kraemer et al., Am. J. Pathol. 128, 446-454 (1987).
It was recently discovered in co-pending U.S. patent application Ser. No. 08/652,327, filed May 22, 1996 and issued as U.S. Pat. No. 5,753,702 on May 19, 1998 and PCT Patent Application No. PCT/US97/08865, and its related national Stage U.S. patent application Ser. No. 09/194,166, the entire contents of which are hereby incorporated by reference, that 16-HETE (16-hydroxyeicosatetraenoic acid) is a component of arachidonic acid metabolism in neutrophils and that 16-HETE is a potent inhibitor of neutrophil adhesion and neutrophil aggregation. It was also disclosed in these applications that 16-HETE when administered alone actually reduces the size of brain infarcts resulting from acute stroke relative to the size of brain infarcts which occur in the absence of a therapeutic. When 16-HETE is administered in combination with clot lysing thrombolytic agents such as tPA, the therapeutic combination actually reduces the size of brain infarcts resulting from acute stroke relative to the size of brain infarcts which occur in a subject suffering from an acute stroke who has been treated only with a thrombolytic agent such as tPA.
The present invention relates to novel analogs of 16-HETE. Some of the analogs of 16-HETE are agonists which maintain the biological activity of 16-HETE but which are more stable and have longer half-lives. The 16-HETE analogs also include 16-HETE antagonists which inhibit the activity of 16-HETE. These antagonists are useful when it is desirable to prevent inhibition of neutrophil activity.
According to one aspect of the invention, compositions are provided. These compositions include the following 16-HETE analog: 
wherein R is selected from the group consisting of xe2x80x94C(O)xe2x80x94Xxe2x80x94SO2xe2x80x94R1, xe2x80x94C(O)xe2x80x94Xxe2x80x94COxe2x80x94R1, xe2x80x94C(O)xe2x80x94Xxe2x80x94C(OH)2xe2x80x94R1, xe2x80x94C(O)xe2x80x94Xxe2x80x94C(NH)2xe2x80x94R1, xe2x80x94C(O)xe2x80x94Xxe2x80x94C(NH2)2xe2x80x94R1, piperonyl,xe2x80x94CN, xe2x80x94ORxe2x80x2, xe2x80x94SRxe2x80x2, xe2x80x94NO2, xe2x80x94NRxe2x80x2Rxe2x80x2, amino acid, xe2x80x94C(O)Rxe2x80x2, xe2x80x94C(S)Rxe2x80x2, xe2x80x94C(O)ORxe2x80x2, xe2x80x94C(S)ORxe2x80x2, xe2x80x94C(O)SR, xe2x80x94C(S)SRxe2x80x2, xe2x80x94C(O)N(Rxe2x80x2)2, xe2x80x94C(O)C(O)Rxe2x80x2, xe2x80x94C(S)C(O)Rxe2x80x2, xe2x80x94C(O)C(S)Rxe2x80x2, xe2x80x94C(S)C(S)Rxe2x80x2, xe2x80x94C(O)C(O)ORxe2x80x2, xe2x80x94C(S)C(O)ORxe2x80x2, xe2x80x94C(O)C(S)ORxe2x80x2, xe2x80x94C(O)C(O)SRxe2x80x2, xe2x80x94C(S)C(S)ORxe2x80x2, xe2x80x94C(S)C(O)SRxe2x80x2, xe2x80x94C(O)C(S)SRxe2x80x2, xe2x80x94C(S)C(S)SRxe2x80x2, xe2x80x94C(O)C(O)N(Rxe2x80x2)2, xe2x80x94C(S)C(O)N(Rxe2x80x2)2, xe2x80x94C(O)C(S)N(Rxe2x80x2)2, or xe2x80x94C(S)C(S)N(Rxe2x80x2)2; wherein X is selected from the group consisting of O, N, and a bond; wherein R1, R2, and R3 each independently is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, aryl, and heteroaryl; wherein each Rxe2x80x2 is (CH2)zxe2x80x94NRxe2x80x3Rxe2x80x3 and wherein Rxe2x80x3 is independently selected from the group consisting of (C1-C6)alkyl, (C1-C6) alkenyl, (C1-C6)alkoxy, (C1-C6) alkynyl, (C6-C20)aryl, (C6-C20) substituted aryl, (C6-C26) alkaryl, substituted (C6-C26)alkaryl, and (C5-C7)heteroaryl.
16-HETE analogs include both agonists and antagonists. In some embodiments the 16-HETE agonists have the following general structure: 
wherein either R1 or R2 is a C3 alkyl and the other is a hydrogen.
In one embodiment R1, R2, and R3 each independently is selected from the group consisting of hydrogen, (C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, and (C1-C6)alkoxy.
In another embodiment the 16-HETE analog has the following structure: 
In a preferred embodiment X is NH and R2 and/or R3 is hydrogen. In another preferred embodiment R3 is a C3 alkyl. Preferably, the 16-HETE analog has the following structure: 
In a preferred embodiment X is O and R2 and/or R3 is hydrogen. In another preferred embodiment R3 is a C3 alkyl.
In other preferred embodiments the 16-HETE analog is one of the following structures: 
In another embodiment the 16-HETE analog is a 16-HETE antagonist having the following general structure: 
wherein R4 and R5 each independently is selected from the group consisting of hydrogen, C1-2 alkyl, C4-6 alkyl, alkenyl, alkynyl, alkoxy, aryl, and heteroaryl and wherein when either R1 or R2 is a C3 alkyl the other is not a hydrogen.
According to another aspect of the invention the 16-HETE analog has the following structure: 
wherein R is selected from the group consisting of xe2x80x94C(O)xe2x80x94Xxe2x80x94SO2xe2x80x94R1, xe2x80x94C(O)xe2x80x94Xxe2x80x94COxe2x80x94R1, xe2x80x94C(O)xe2x80x94Xxe2x80x94C(OH)2xe2x80x94R1, xe2x80x94C(O)xe2x80x94Xxe2x80x94C(NH)2xe2x80x94R1, xe2x80x94C(O)xe2x80x94Xxe2x80x94C(NH2)2xe2x80x94R1, piperonyl,xe2x80x94CN, xe2x80x94ORxe2x80x2, xe2x80x94SRxe2x80x2, xe2x80x94NO2, xe2x80x94NRxe2x80x2Rxe2x80x2, amino acid, xe2x80x94C(O)Rxe2x80x2, xe2x80x94C(S)Rxe2x80x2, xe2x80x94C(O)ORxe2x80x2, xe2x80x94C(S)ORxe2x80x2, xe2x80x94C(O)SR, xe2x80x94C(S)SRxe2x80x2, xe2x80x94C(O)N(Rxe2x80x2)2, xe2x80x94C(O)C(O)Rxe2x80x2, xe2x80x94C(S)C(O)Rxe2x80x2, xe2x80x94C(O)C(S)Rxe2x80x2, xe2x80x94C(S)C(S)Rxe2x80x2, xe2x80x94C(O)C(O)ORxe2x80x2, xe2x80x94C(S)C(O)ORxe2x80x2, xe2x80x94C(O)C(S)ORxe2x80x2, xe2x80x94C(O)C(O)SRxe2x80x2, xe2x80x94C(S)C(S)ORxe2x80x2, xe2x80x94C(S)C(O)SRxe2x80x2, xe2x80x94C(O)C(S)SRxe2x80x2, xe2x80x94C(S)C(S)SRxe2x80x2, xe2x80x94C(O)C(O)N(Rxe2x80x2)2, xe2x80x94C(S)C(O)N(Rxe2x80x2)2, xe2x80x94C(O)C(S)N(Rxe2x80x2)2, or xe2x80x94C(S)C(S)N(Rxe2x80x2)2; wherein X is selected from the group consisting of O, NH, and a bond; wherein R1, R2, and R3 each independently is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, aryl, and heteroaryl; wherein each Rxe2x80x2 is (CH2), xe2x80x94NRxe2x80x3Rxe2x80x3 and wherein Rxe2x80x3 is independently selected from the group consisting of (C1-C6) alkyl, (C1-C6) alkenyl, (C1-C6) alkoxy, (C1-C6) alkynyl, (C6-C20) aryl, (C6-C20) substituted aryl, (C6-C26) alkaryl, substituted (C6-C26) alkaryl, and (C5-C7) heteroaryl.
In one embodiment R1, R2, and R3 each independently is selected from the group consisting of hydrogen, (C1-C6) alkyl, (C1-C6) alkenyl, (C1-C6) alkynyl, and (C1-C6) alkoxy.
In another embodiment the 16-HETE analog has the following structure: 
In a preferred embodiment X is NH and R2 and/or R3 is hydrogen. In another preferred embodiment R3 is a C3 alkyl. Preferably, the 16-HETE analog has the following structure: 
In a preferred embodiment X is O and R2 and/or R3 is hydrogen. In another preferred embodiment R3 is a C3 alkyl. In another preferred embodiment the 16-HETE analog has the following structure: 
Several diseases or conditions are characterized by excessive inflammation associated with neutrophil adhesion and neutrophil aggregation. The present invention provides compositions for inhibiting neutrophil adhesion and neutrophil aggregation as well as for treating subjects having diseases or conditions characterized by excessive inflammation associated with neutrophil adhesion and neutrophil aggregation. In some embodiments the composition is a pharmaceutical composition of a 16-HETE agonist. In these embodiments the 16-HETE agonist is present in a therapeutically effective amount for treating an adverse medical condition mediated by neutrophil adhesion and/or neutrophil aggregation. In an embodiment, the pharmaceutical preparation of the invention includes other therapeutic agents for treating the adverse medical condition mediated by neutrophil adhesion and/or neutrophil aggregation. For instance when the medical condition is an inflammatory disease the other therapeutic agents are agents for treating an inflammatory disease or condition. According to particular embodiments of the invention the therapeutic agent for treating the inflammatory disease or condition is selected from the group consisting of antibiotics, such as tetracycline antibiotics, aminoglycosides, macrolides, lincomycins, penicillanic acid (6-APA)-derivatives having 6xcex2-acylamino groups, cephalosporanic acid (7-ACA)-derivatives having 7xcex2-acylamino groups, penicillanic acid, xcex2-lactam antibiotics of the clavam, penem and carbapenen type, and antibiotics of the bicozamycin, novobiocin, chloramphenicol or thiamphenicol, rifampicin, fosfomycin, colistin and vancomycin and antiphlogistics, such as glucocorticoids, immunosuppressive agents, penicillamine, hydroxychloroquine, and nonsteroidal inflammation-inhibitors.
According to another embodiment, the pharmaceutical preparation includes therapeutic agents for treating an ischemic disease or condition. Preferably the therapeutic agent is selected from the group consisting of: anticoagulation agents, such as heparin, warfarin, coumadin, dicumarol, phenprocoumon, acenocoumarol, ethyl biscoumacetate, and indandione derivatives; antiplatelet agents, such as aspirin, thienopyridine, dipyridamole and sulfinpyrazone; and thrombolytic agents, such as plasminogen, a2-antiplasmin, streptokinase, antistreplase, tissue plasminogen activator, and urokinase.
According to one preferred embodiment of the invention, the pharmaceutical preparation includes the 16-HETE agonist of the invention together with a thrombolytic agent. The pharmaceutical preparation is formulated for intravenous administration in one embodiment. In another embodiment the thrombolytic agent is recombinant tPA. In another embodiment the thrombolytic agent is a modified tPA. In one embodiment the modified tPA is T103N, N117Q, KHRR (296-299) AAAA tPA. In another embodiment the modified tPA is a vampire bat tPA selected from the group consisting of Bat-Pa(H), Bat-Pa(I), and Bat-PA(L).
The invention also provides a composition of 16-HETE analog for use as a medicament.
The invention also provides a composition of 16-HETE agonist for use in the manufacture of a medicament for the treatment of adverse medical conditions mediated by neutrophil adhesion and/or neutrophil aggregation. In another embodiment the invention provides a composition of 16-HETE agonist for use in the manufacture of a medicament for the treatment of an inflammatory disease or condition. In another embodiment the invention provides a composition of 16-HETE agonist for use in the manufacture of a medicament for the treatment of an ischemic disease or condition, including but not limited to, acute stroke.
The invention also encompasses methods of treatment. According to another aspect of the invention the 16-HETE agonist may be administered to a subject in conjunction with other drugs for treating an adverse medical condition mediated by neutrophil adhesion and/or neutrophil aggregation. In one embodiment the condition mediated by neutrophil adhesion and/or neutrophil aggregation is an inflammatory disease or condition. According to particular embodiments, the inflammation is characteristic of or results from meningitis, cerebral edema, arthritis, nephritis, adult respiratory distress syndrome, pancreatitis, myositis, neuritis, connective tissue disease, phlebitis, arteritis, vasculitis, allergy, anaphylaxis, gout, ulcerative colitis, and/or ehrlichiosis. In one embodiment the method also includes the step of administering to the subject a therapeutic agent other than and in addition to 16-HETE agonist for treating the inflammatory condition. According to another embodiment, the condition mediated by neutrophil adhesion and/or neutrophil aggregation is an ischemic disease or condition. Preferably the ischemic condition is selected from the group consisting of a stroke and a myocardial infarction. In one embodiment the 16-HETE agonist is administered to a subject having an ischemic disease or condition, in conjunction with a thrombolytic agent.
The 16-HETE agonist may be administered by any known method of drug delivery. Preferably the 16-HETE agonist is administered orally or intravenously.
One aspect of the invention is directed to a method for inhibiting neutrophil adhesion and neutrophil aggregation. The method involves contacting neutrophils with a 16-HETE agonist in situ in an amount effective to inhibit neutrophil adhesion and neutrophil aggregation. This aspect of the invention may be applied in vitro or in vivo to inhibit neutrophil adhesion and neutrophil aggregation at a desired time.
In one aspect, the invention is a method for treating thromboembolic stroke. The method involves administering to a subject experiencing an acute thromboembolic stroke 16-HETE agonist in combination with a thrombolytic agent in an amount effective to reduce brain injury which would otherwise occur as a result of the stroke. In one embodiment, the 16-HETE agonist is 16(R)-HETE agonist and the thrombolytic agent is tPA. In another embodiment the 16-HETE agonist and thrombolytic agent are administered to the subject within a first 2-6 hours after the subject experienced the thromboembolic stroke. In one embodiment the 16-HETE agonist is administered in an amount between 0.5 and 20 mg/kg per minute. In a preferred embodiment the 16-HETE agonist is 16(R)-HETE agonist and is administered in an amount of 1.0 mg/kg per minute. In one embodiment the thrombolytic agent is administered in an amount between 0.05 mg/kg and 1.5 mg/kg. In a preferred embodiment the thrombolytic agent is tPA and is administered in an amount of 0.9 mg/kg. In one embodiment the thrombolytic agent is recombinant tPA. In another embodiment the thrombolytic agent is a modified tPA. In one embodiment the modified tPA is T103N, N117Q, KHRR (296-299) AAAA tPA. In another embodiment the modified tPA is a vampire bat tPA selected from the group consisting of Bat-PA(H), Bat-Pa(I), and Bat-PA(L). In yet another embodiment the modified tPA is C84S tPA.
According to another aspect of the invention a method of inhibiting leukotriene production in a neutrophil is provided. Leukotriene is an arachidonic acid metabolite that is a potent neutrophil chemoattractant and pro-aggregant. It was discovered according to the invention that 16-HETE and agonists thereof inhibit leukotriene production in neutrophils. The method includes the step of administering to a neutrophil, 16-HETE or an agonist thereof in an amount effective to inhibit leukotriene production.
The invention in another aspect is a method of inhibiting leukotriene production in a subject having a condition mediated by leukotriene activity. The method involves the step of administering to said subject having a condition mediated by leukotriene activity a 16-HETE or an agonist thereof in an amount effective to inhibit leukotriene production. In a preferred embodiment the leukotriene is leukotriene B4. In another embodiment the leukotriene production is inhibited in neutrophils.
In one embodiment the condition mediated by leukotriene activity is selected from the group consisting of arthritis, rheumatoid arthritis, osteoarthritis, allergic rhinitis, psoriasis, dermatitis, ischemic induced myocardial injury, reperfusion injury, gout, asthma, adult respiratory distress syndrome, atherosclerosis, inflammatory disease, stroke, spinal cord injury, and traumatic brain injury. Preferably the condition mediated by leukotriene activity is an inflammatory disease.
Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each method and product.
These and other aspects of the invention, as well as various advantages and utilities, will be more apparent with reference to the detailed description of the preferred embodiments.